Alkaline phosphatase from Microorganism

1. Features
2. Property
Description Alkaline phosphatase (E.C.
Reaction Orthophosphoric-monoester + H₂O → alcohol + Orthophosphate
Origin Microorganism (Bacterium)
Specific activity ≧6,000 U/mg-protein (37°C・pH=9.8, pNPP is used as a substrate)
Molecular weight Approx. 100,000 (by gel filtration)
Optimum pH Approx. 9.8
pH stability 6.4 – 9.0 (25°C, 24hr)
Thermalstability ≦60°C (pH7.5,1hr)
Stabilizer Mg²⁺、Zn²⁺
Available substrates p-Nitrophenyl phosphate, Dioxietane-based substrates, Acridan-based
substrates, 5-Bromo-4-Chloro-3-Indoxyl Phosphate (BCIP),
4-Methylumbelliferyl Phosphate, etc.
Stability Stable at 4°

3. The data in ELISA test


4. Alkaline phosphatase ASSAY


Alkaline phosphatase

p-Nitorphenylphosphate (pNPP)+H₂O                                                   P-Nitrophenol + Pi
The appearance of p-Nitrophenol is measured at 405nm by spectrophotometry .

Unit definition:

One unit causes the formation of one micromole of p-Nitrophenol per minute under the conditions described below .


A. Diethanolamine buffer 1M [Dilute 9.66ml of diethanolamine (MW=105.14) in 60ml H₂O, add 5ml of 0.1M MgCl₂ and, after adjusting the pH at 37°C to 9.8 with 2N HCl, fill up to 100ml with H₂O]
B. pNPP solution 0.674M [2.5g p-Nitorphenylphosphate disodium salt (MW=371.16) / 10ml Diethanolamine buffer (A)] (Prepare freshly)
C. Enzyme diluent 30mM Triethanolamine, 1mM MgCl₂ , 0.1mM ZnCl. 0.1% Triton X-100 pH=7.6


Concentration in assay mixture
Diehtanolamine 0.97 M
P-Nitrophenylphosphate 11 mM
MgCl₂ 4.8 mM

1. Prepare the following working solution (30.5ml) in a brownish bottle and store on ice. (Prepare freshly)

30ml Diethanolamine buffer (A)
0.5ml pNPP solution (B)

2. Pipette 3.0ml of working solution into a cuvette (d=1.0cm) and equilibrate at 37°C for about 5minutes .

3. Add 0.1ml of the enzyme solution* and mix by gentle inversion.

4. Record the increase of optical density at 405nm against water for 3 to 5 minutes in a spectrophotometer
thermostated at 37°C, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔODtest) . At the same time , measure the blank rate (ΔODblank) by the same method as test except that the enzyme diluent (C) is added instead of the enzyme solution .

* Dilute to 0.1 – 0.3 U/ml with ice cold enzyme diluent (C), immediately before assay .


Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test-ΔOD blank)×Vt×df

Volume activity (U/ml) =                                                               = ΔOD/min×1.676×df


: Total volume (3.1ml)
: Sample volume (0.1ml)
: Millimolar extinction coefficient of p-Nitrophenol under the assay conditions (㎠/micromole)
: Light path length (cm)
: Dilution factor

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